Hydroxyphenylpyruvate dioxygenases are enzymes which catalyze the reaction of conversion of para-hydroxyphenylpyruvate (HPP) to homogentisate. This reaction takes place in the presence of iron (Fe2+) in the presence of oxygen (N. P. Crouch et al., Tetrahedron, 53, 20, 6993-7010, 1997).
Certain molecules which inhibit this enzyme are, moreover, known, which attach to the enzyme so as to inhibit the conversion of HPP to homogentisate. Some of these molecules have found a use as herbicides, insofar as inhibition of the reaction in plants leads to a bleaching of the leaves of the treated plants and to the death of said plants (K. E. Pallett et al., 1997 Pestic. Sci. 50 83-84). Such herbicides having HPPD as the target, described in the state of the art, are in particular isoxazoles (EP 418 175, EP 470 856, EP 487 352, EP 527 036, EP 560 482, EP 682,659, U.S. Pat. No. 5,424,276), in particular isoxaflutole, a herbicide selective for maize, diketonitriles (EP 496 630, EP 496 631), in particular 2-cyano-3-cyclopropyl-1-(2-SO2CH3-4-CF3-pheny-1) propane-1,3-dione and 2-cyano-3-cyclopropyl-1-(2-SO2CH3-4-2,3-Cl2phenyl))propane-1,3-dione, triketones (EP 625 505, EP 625 508, U.S. Pat. No. 5,506,195), in particular sulcotrione or mesotrione, or else pyrazolinates.
Assays to confirm that HPPD is indeed the target for diketonitriles (DKNS) and to demonstrate that HPPD is, at least at certain doses, the only target for diketonitriles, were carried out in the laboratory by germinating Arabidopsis seeds on three types of medium under sterile conditions in vitro:
1 Murashige and Skoog medium (T. Murashige and F. Skoog, 1962. A revised medium for a rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 15, 473-479), control experiment for germination
2 MS medium plus DKN at a dose of 1 ppm
3 MS medium plus DKN at the same dose+homogentisate at a concentration of 5 mM.
It is very clear that, on medium 1, germination occurs normally, each plantlet developing two cotyledons which are clearly green. Development then takes place normally. On medium 2, germination occurs, but the plantlet which emerges is white, the two cotyledons exhibiting no pigmentation. The plantlets then die in a few days. On medium 3, germination occurs normally, the cotyledons are clearly green. The plants develop, but very rapidly, the amount of homogentisate in the medium decreasing, the first symptoms of bleaching appear and plant growth stops, they end up dying as in the assay carried out on medium No. 2.
This makes it possible to confirm that HPPD is clearly the target for DKNs in plants and that it appears to be the only target. This also shows that homogentisate is transported from the culture medium to the cell site where it is necessary for correct functioning of the cell and survival of the plant.
Three strategies are currently available to make plants tolerant to herbicides, (1) detoxification of the herbicide with an enzyme which converts the herbicide, or its active metabolite, to nontoxic degradation products, such as, for example, the enzymes for tolerance to bromoxynil or to basta (EP 242 236, EP 337 899); (2) mutation of the target enzyme to a functional enzyme less sensitive to the herbicide, or its active metabolite, such as, for example, the enzymes for tolerance to glyphosate (EP 293 356, S. R. Padgette et al., J. Biol. Chem., 266, 33, 1991); or (3) overexpression of the sensitive enzyme, so as to produce in the plant amounts of target enzyme which are sufficient with regard to the kinetic constants of this enzyme with respect to the herbicide in order to have sufficient functional enzyme, despite the presence of its inhibitor.
This third strategy which has been described for successfully obtaining plants tolerant to HPPD inhibitors (WO 96/38567), it being understood that, for the first time, a strategy of simple overexpression of the sensitive (nonmutated) target enzyme, was used successfully to impart on plants tolerance at an agronomic level to a herbicide. The identification of HPPDs mutated in the C-terminal portion which exhibit improved tolerance to HPPD inhibitors has made it possible to obtain an improvement in the tolerance of plants using the second strategy (WO 99/24585).